LOCUS       sequence-73302-e        7306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7306)
  AUTHORS   Stanton BC, Nielsen AA, Tamsir A, Clancy K, Peterson T, Voigt CA
  TITLE     Genomic mining of prokaryotic repressors for orthogonal logic gates.
  JOURNAL   Nat Chem Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411.
  PUBMED    24316737
REFERENCE   2  (bases 1 to 7306)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411."
COMMENT     Sequence Label: pRF-BetI
FEATURES             Location/Qualifiers
     source          1..7306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(3..8,9..24,25..31)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..56
                     /label=lac operator (symmetric)
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     terminator      704..775
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      791..818
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             896..907
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(914..916,917..919,920..1633)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(959..980)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1220..1239)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1567..1588
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1673..1690)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1843..1929
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2021..2048
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2067..2158
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2159..2227,2228..3019)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3061..3516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3148..3167)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4347..4364)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4481..5026)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4561..4580)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5098..5117
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(6092..7174)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7136..7155
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattgcta
       61 cgacttgctc atttgacaga ggataactac tagtgccgaa actgggtatg cagagcattc
      121 gtcgtcgtca gctgattgat gcaaccctgg aagcaattaa tgaagttggt atgcatgatg
      181 caaccattgc acagattgca cgtcgtgccg gtgttagcac cggtattatt agccattatt
      241 tccgcgataa aaacggtctg ctggaagcaa ccatgcgtga tattaccagc cagctgcgtg
      301 atgcagttct gaatcgtctg catgcactgc cgcagggtag cgcagaacag cgtctgcagg
      361 caattgttgg tggtaatttt gatgaaaccc aggttagcag cgcagcaatg aaagcatggc
      421 tggcattttg ggcaagcagc atgcatcagc cgatgctgta tcgtctgcag caggttagca
      481 gtcgtcgtct gctgagcaat ctggttagcg aatttcgtcg tgaactgcct cgtgaacagg
      541 cacaagaggc aggttatggt ctggcagcac tgattgatgg tctgtggctg cgtgcagcac
      601 tgagcggtaa accgctggat aaaacccgtg caaatagcct gacccgtcat tttatcaccc
      661 agcatctgcc gaccgattaa tcgtaaggcg agcccccagg catcaaataa aacgaaaggc
      721 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
      781 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataataccg tcattcatgg
      841 attcgttacc aattgacaat tgattggacg ttcaatataa tgctagctac tagagaaaga
      901 ggagaaatac tagatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
      961 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1021 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1081 gccctggccc accctcgtga ccaccttcgg ctacggcctg caatgcttcg cccgctaccc
     1141 cgaccacatg aagctgcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1201 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1261 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1321 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     1381 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     1441 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1501 gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg
     1561 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1621 gctgtacaag taataagctt ggctgttttg gcggatgaga gaagattttc agcctgatac
     1681 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg
     1741 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
     1801 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
     1861 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     1921 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     1981 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
     2041 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta
     2101 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagactat
     2161 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2221 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2281 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2341 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2401 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2461 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2521 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2581 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2641 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     2701 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     2761 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     2821 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     2881 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     2941 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3001 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3061 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3121 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3181 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3241 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     3301 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3361 gactcttgtt ccaaacttga acaacactca accctatctc gggctattct tttgatttat
     3421 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3481 acgcgaattt taacaaaata ttaacgttta caatttaaaa ggatctaggt gaagatcctt
     3541 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3601 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3661 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3721 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3781 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3841 ctgctaatcc tgttaccagt caggcatttg agaagcacac ggtcacactg cttccggtag
     3901 tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac
     3961 cgacgaccgg gtcgaatttg ctttcgaatt tctgccattc atccgcttat tatcacttat
     4021 tcaggcgtag caccaggcgt ttaagggcac caataactgc cttaaaaaaa ttacgccccg
     4081 ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca
     4141 tcacagacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta
     4201 taatatttgc ccatggctag cggagtgtat actggcttac tatgttggca ctgatgaggg
     4261 tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg caccggtgcg tcagcagaat
     4321 atgtgataca ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga
     4381 ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag
     4441 atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc
     4501 ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat
     4561 aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc
     4621 ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc
     4681 agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc
     4741 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa
     4801 gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc
     4861 cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc
     4921 tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt
     4981 ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat
     5041 taatcagata aaatatttgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     5101 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     5161 gcgtccggcg tagaggatct gctcatgttt gacagcttat catcgatgca taatgtgcct
     5221 gtcaaatgga cgaagcaggg attctgcaaa ccctatgcta ctccctcgag ccgtcaattg
     5281 tctgattcgt taccaattat taatttttaa agtatgggca atcaattgct cctgttaaaa
     5341 ttgctttaga aatactttgg cagcggtttg ttgtattgag tttcatttgc gcattggtta
     5401 aatggaaagt gacagtacgc tcactgcaac ctaatatttt tgaaatatcc caagagcttt
     5461 ttccttcgca tgcccacgct aaacattctt tttctctttt ggttaaatcg ttgtttgatt
     5521 tattatttgc tatatttatt tttcgataat tatcaactag agaaggaaca attaatggta
     5581 tgttcataca cgcatgtaaa aataaactat ctatatagtt gtctttttct gaatgtgcaa
     5641 aactaagcat tccgaagcca ttgttagccg tatgaatagg gaaactaaac ccagtgataa
     5701 gacctgatgt tttcgcttct ttaattacat ttggagattt tttatttaca gcattgtttt
     5761 caaatatatt ccaattaatt ggtgaatgat tggagttaga ataatctact ataggatcat
     5821 attttattaa attagcgtca tcataatatt gcctccattt tttagggtaa ttatctagga
     5881 ttgaaatatc agatttaacc atagaatgag gataaatgat cgcgagtaaa taatattcac
     5941 aatgtaccat tttagtcata tcagataagc attgattaat atcattattg cttctacaag
     6001 ctttaatttt attaattatt ctgtatgtgt cgtcggcatt tatgtttttc atcgaggcct
     6061 ttcctgtgtg acccgctgga ataggatctt atcactgccc gctttccagt cgggaaacct
     6121 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6181 gcgccagggt ggtttttctt ttcaccagtg agactggcaa cagctgattg cccttcaccg
     6241 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     6301 cctgtttgat ggtggttaac ggcgggatat aacatgagct atcttcggta tcgtcgtatc
     6361 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     6421 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     6481 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     6541 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     6601 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     6661 ccacgcccag tcgcgtaccg tcctcatggg agaaaataat actgttgatg ggtgtctggt
     6721 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     6781 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     6841 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     6901 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     6961 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     7021 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     7081 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     7141 actctgcgac atcgtataac gttactggtt tcatattcac caccctgaat tgactctctt
     7201 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggcg cgccgcttcg
     7261 tcaggccaca tagctttctt gttctgatcg gaacgatcgt tggctg
//
